The overall goal of this grant over the years has been to characterize the detailed mechanism of action of various physiologically important forms of phospholipase A2 (PLA2). During the course of these studies, it has become apparent that the activity of this superfamily of enzymes depends critically on the interaction of the proteins with large lipid aggregates, where the orientation of the enzyme with respect to the plane of the lipid-water interface can have a dramatic effect on activity. The nature of this interaction has been difficult to explore because of the fact that it represents the interaction of two large macromolecules. This has presented challenges for traditional NMR and X-ray crystallographic studies. The activity of many of these enzymes increases when the enzyme is at the lipid-water interface. This activation is due in part to changes in enzyme-lipid orientation and to conformational changes in the enzyme. This renewal application will extend our current studies on the human Ca2+-independent Group VIA iPLA2 (GVIA iPLA2) and the human lipoprotein-associated PLA2 / PAF (platelet activating factor) acetyl hydrolase Group VIIA Lp-PLA2 (GVIIA Lp-PLA2). The GVIA iPLA2 is responsible for remodeling of membrane phospholipids in cells and plays critical roles in the cellular regulation of several diseases. We will determine exactly how it interacts with membranes. The GVIIA Lp-PLA2 is found associated with lipoproteins, both LDL and HDL, and is implicated in the turnover of oxidized phospholipids, as well as PAF, and has been implicated in cardiovascular disease. Along with traditional biochemical, molecular biological, and kinetic approaches, we will employ amide hydrogen/deuterium exchange-mass spectrometry (DXMS). It is rapidly becoming clear that this technique can tackle many structural questions about how proteins act in solution that cannot be addressed easily by NMR or X-ray crystallography. We will apply the DXMS technique to explore the interactions of these enzymes with large lipid interfaces and specific potent inhibitors as we have done earlier under this grant with the GIA sPLA2 and GIVA cPLA2. We also will use surface plasmon resonance and our detailed surface dilution kinetic model to study the functional aspects of these questions. This work will generate important widely applicable information on how soluble enzymes interact with lipid-water interfaces.